4.6 | Introduction of fluorescence-labeled PCNDs into
cells
To microscopically observe the intracellular accumulation of PCNDs,
PCNDs were modified with fluorescence-labeled 9E5 and
fluorescence-labeled PEG-lipid. 9E5 was prepared using 9E5-expressing
hybridoma cells[31,32] and biotinylated by
reacting with biotin-NHS (13 eq), which was synthesized as previously
reported[39]. After purification with a gel
filtration column, biotinylated 9E5 was fluorescently labeled by
allowing it to react with Alexa647-NHS (10 eq). Biotinylated and
fluorescently labeled 9E5 was purified by gel filtration and then was
conjugated with PCNDs, as previously
reported[30,31]. Here, PCNDs were prepared using
DPPC, DPPE-PEG-OMe, DPPE-PEG-biotin, and DPPE-PEG-TAMRA. Biotinylated
and fluorescently labeled 9E5 was mixed with NeutrAvidin for 15 min at
room temperature to prepare the 9E5-NeutrAvidin conjugate. PCNDs were
suspended in 9E5-NeutrAvidin solution and incubated for 30 min on ice
for modification with 9E5. After centrifugation at 3000 × g for 5
min, the PCND pellet was washed with cold PBS and suspended in
RPMI-based culture medium. DLD1 cells were treated with 9E5-conjugated
PCND suspension for 3 h under culture conditions. After washing the dish
surface, the cells were observed using a confocal laser-scanning
microscope (LSM 510 META; Carl Zeiss, Germany) equipped with a 20×
objective lens. The fluorescence of TAMRA (Em: 560/610 nm) and AF647
(Em:650/680 nm) was observed using excitation lasers at wavelengths of
552 and 633 nm, respectively.